PROJECT SUMMARY ABSTRACT Although the pathological hallmarks of AD, such as axonal transport defects, synaptic loss, and selective neuronal death, are well-characterized, the underlying mechanisms that cause AD are largely unknown, thereby making it difficult to design effective therapies. Although the majority of AD patients are sporadic, several genetic risk variants have been identified. Variation in the Apoplioprotein E (ApoE) gene, a lipoprotein transporter involved in cholesterol metabolism, has been identified as the most powerful and prevalent of these risk factors. Specifically, compared to the referent variant ?3 allele, individuals with one or two ?4 alleles have a 3- or 12-fold higher probability of developing AD, respectively. On the other hand, the ?2 allele reduces patient risk for AD by nearly 50%. Amyloid-dependent and -independent mechanisms have been postulated to explain the risk inducing ApoE4 effect, but the molecular and cellular mechanisms by which ApoE2 reduces AD disease risk remain unclear. Current studies to examine the neuroprotective effect of ApoE2 have been limited to (i) rodent models, which while have provided valuable information in understanding AD, but do not recapitulate all aspects of the human disease and (ii) neuronal cells from cadaveric tissue samples which are limited in supply and rapidly loose disease-related phenotypes upon extensive in vitro culture. With hiPSC technology, it is possible to obtain a fully differentiated cell type (such as a skin cell) from an AD patient and reprogram it back into a cell type that is capable of differentiating into all of the cell types of the mature, adult body (such as neural cells of the cortex). Although we have used AD hiPSC-derived neural cells to study this disease in a simplified and accessible system, our study of the effect of ApoE variants on the manifestation of AD-related phenotypes has been confounded by the variance that exists between individual patient genomes. To that end, we will use our collective experience in stem cell bioengineering, neurodegenerative disease modeling, and genome editing to elucidate potential genetic, molecular, and cellular mechanisms by which ApoE2 protects against AD onset and age-related progression. In the first aim of this proposal, we will use a novel adeno-associated virus (AAV) and CRISPR/Cas9 dual approach to efficiently generate isogenic hiPSC lines that only differ with respect to their ApoE genotype and not genetic background. In the second aim, subsequent phenotypic analysis of 3-D cortical neural cultures derived from these isogenic lines will reveal the (i) direct effect of the presence of an ?2 allele on the manifestation of AD-related phenotypes and (ii) potential signaling pathways and transcriptional targets that are independently influence by ApoE genotype and disease status. Overall, the ability to identify definitive relationships between ApoE genotype and AD-related phenotypes will have a significant translational impact on the design of molecularly targeted therapies to treat the many patients suffering from AD.